ASSAY OF ALPHA-AMYLASE. using RED-STARCH S-RSTAR 03/06

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1 ASSAY OF ALPHA-AMYLASE using RED-STARCH S-RSTAR 03/06 Megazyme International Ireland 2006

2 PRINCIPLE: This assay procedure employing Red Starch is relatively specific for α-amylase, however, the substrate is also hydrolysed at a slow rate by pullulanase (limit-dextrinase). On incubation of Red-Starch with α-amylase the substrate is depolymerised by an endo-mechanism to produce low-molecular weight dyed fragments which remain in solution on addition of ethanol to the reaction mixture. High molecular weight material is removed by centrifugation, and the colour of the supernatant is measured at 510 nm. α-amylase in the assay solution is determined by reference to a standard curve, through which, absorbance at 510 nm is converted to Ceralpha Units (CU). The Ceralpha method is the only procedure which employs a well defined substrate (ie. end-blocked p-nitrophenyl maltoheptaoside). Factors to convert Ceralpha Units to International units, Dextrinising Units, SKB Units or Farrand Units can be obtained by reference to the Ceralpha Kit (K-CERA) Booklet. SUBSTRATE: The substrate is partially depolymerised starch which is dyed with Procion Red MX-5B to an extent of approx. one dye molecule per 30 sugar residues. Add 1.0 g of powdered substrate to 50 ml of 0.5 M potassium chloride solution and stir vigorously at 60 C until it completely dissolves (about 10 min). Store the substrate at 4 C between use; it is stable for several months in the liquid form if it is stored in a well sealed glass bottle, overlain with a few drops of toluene to prevent microbial growth. BUFFERS: A. Buffer A (for cereal and fungal α amylase) Malic acid (Sigma M-0875; 1M) Sodium hydroxide Calcium chloride. 2H 2 O (40 mm) Sodium azide (Sigma S-2002; 0.1%) g/l 70.0 g/l 5.9 g/l 1.0 g/l Add the malic acid and sodium hydroxide to 800 ml of distilled water and dissolve by stirring. Allow the solution to cool to room temperature and add the calcium chloride. Adjust the ph to 5.4 by dropwise addition of sodium hydroxide (4 M) or HCl (4 M). Then add the sodium azide. Adjust volume to 1 Litre. Store at room temperature. For use, dilute 100 ml of this concentrated buffer solution to 1 Litre of distilled water. 1

3 B. Buffer B (for Bacillus sp. α-amylase). Maleic acid (Sigma M-0375; 0.1 M) Calcium chloride. 2H 2 O (2 mm) Sodium azide (Sigma S-2002; 0.01%) 23.2 g/2 L 0.6 g/2 L 0.2 g/2 L Add the maleic acid to 1.6 Litre of distilled water and adjust the ph to 6.5 with 4 M sodium hydroxide (160 g/l). Add the sodium azide and adjust the volume to 2 litres. Store at room temperature between use. Use this buffer directly without further dilution. CAUTION Dissolve the reagents and adjust the ph to 5.4 or 6.5 before adding the sodium azide. Adding sodium azide to an acidic solution results in the release of a poisonous gas. Powdered malic and maleic acids are irritants, and thus should be handled with due care. ENZYME PREPARATION: A. Malt Flours: 1. Mill malt (20 g sample) to pass a 0.5 mm screen. 2. Accurately weigh 1.0 g malt flour into a 50 ml volumetric flask. 3. To the volumetric flask add Buffer A and adjust to volume. 4. Allow the enzyme to extract for min at room temperature, with occasional stirring. 5. Filter an aliquot of the solution through a Whatman No.1 (9 cm) filter paper, or centrifuge at 1,000 g for 10 min. 6. Dilute 1.0 ml of the filtrate with 9.0 ml of Buffer A or B (ie.10-fold). Assay activity within 2 hours. 2

4 B. Microbial Preparations: Liquid preparations: 1. Add 1 ml of liquid enzyme preparation (using a positive displacement dispenser) to buffer A or B (49 ml, ph 5.4 or 6.5) and mix thoroughly. This is termed the Original Extract. 2. Dilute an aliquot of Original Extract 10-fold by adding 1.0 ml to 9.0 ml of the appropriate buffer (A or B) and mix thoroughly. Repeat this step until a dilution suitable for assay is obtained. For example, for the industrial enzyme preparation, Bacterial Alpha-Amylase (from Novo Nordisk) a dilution of the Original Extract of approximately 500-fold is required. Powder preparations: 1. Add 1 g of enzyme powder preparation to 50 ml of buffer A or B (ph 5.4 or 6.5) and gently stir the slurry over a period of about 15 min or until the sample is completely dispersed or dissolved. 2. Clarify this solution (the Original Extract) by centrifugation (1,000 g, 10 min) or filtration through Whatman No.1 (9 cm) filter circles. 3. Dilute an aliquot of this solution 10-fold by adding 1.0 ml to 9.0 ml of appropriate buffer (A or B) and mix thoroughly. Repeat this step until a dilution suitable for assay is obtained [i.e. 2,000-fold for Fungamyl powder (1767 FAU; Novo Nordisk)]. ASSAY PROCEDURE: Malt and microbial preparations: 1. Dispense 0.5 ml aliquots of Red Starch solution (2% w/v, in 0.5 M KCl) into test tubes and pre-incubate the tubes and contents at 40 C for 5 min. 2. Pre-incubate buffered malt or microbial preparation extract at 40 C for 5 min. 3. To each tube containing 0.5 ml of Red Starch solution, add 1.0 ml of pre-equilibrated (and suitably diluted) microbial enzyme or malt extract directly to the bottom of the tube. Incubate at 40 C for exactly 10 min (from time of addition). Terminate the reaction by adding 2.5 ml of ethanol (95% v/v) with vigorous stirring for 10 sec on a vortex mixer. Allow the reaction tubes to equilibrate to room temperature for 10 min and then centrifuge at 1,000 g for 10 min. 3

5 4. Pour the supernatant solution directly from the centrifuge tube into a spectrophotometer cuvette and measure the absorbance of reaction blank and reaction solutions at 510 nm against distilled water. Activity is determined by reference to a standard curve. The reaction blank is prepared by adding ethanol to the Red Starch substrate before addition of the enzyme. Usually, only a single blank is required with each set of determinations. STANDARD CURVE: Standard curves for malt, fungal and bacterial α-amylases are shown in Figs. 1-3, respectively. Enzyme activity is standardised against the Ceralpha method (employing endblocked p-nitrophenyl maltoheptaoside in the presence of excess quantities of thermostable α-glucosidase). One Unit of activity is defined as the amount of enzyme required to release one μmole of p-nitrophenol from blocked p-nitrophenyl maltoheptaoside per minute under the defined assay conditions of temperature and ph. Absorbance, 510 nm α Amylase activity, ceralpha m-u/assay (ie./1.0 ml) Figure 1. Standard curve relating activity of malted barley α-amylase on Red Starch (lot 90101) (as absorbance at 510 nm) to Ceralpha Units (milli-units/assay). 4

6 α-amylase activity, ceralpha m-units/assay (ie./1.0 ml) milli-ceralpha U/mL = 897 x Absorbance + 17 Absorbance, 510 nm Figure 2. Standard curve relating activity of A. niger α-amylase on Red Starch (lot 90101) (as absorbance at 510 nm) to Ceralpha Units (milli-units/assay). α-amylase activity, ceralpha m-units/assay (ie./1.0 ml) milli-ceralpha U/mL = 203 x Absorbance + 5 Absorbance, 510 nm Figure 3. Standard curve relating activity of B. subtilis α-amylase on Red Starch (lot 90101) (as absorbance at 510 nm) to Ceralpha Units (milli-units/assay). 5

7 CALCULATIONS: Units/mL of original solution = milliunits per assay (ie. per 1.0 ml) x 50 x 1 x Dilution 1000 where: milliunits per assay is determined by reference to the Standard Curve 50 = initial extn. or dilution volume (e.g. 1 g per 50 ml). 1 = conversion from milliunits to Units Dilution = further dilution of the original enzyme extract ( = 10-fold for malt extracts). 6

8 Megazyme International Ireland, Bray Business Park, Bray, Co.Wicklow, IRELAND. Telephone: (353.1) Facsimile: (353.1) Internet: www. megazyme.com WITHOUT GUARANTEE The information contained in this booklet is, to the best of our knowledge, true and accurate, but since the conditions of use are beyond our control, no warranty is given or is implied in respect of any recommendation or suggestions which may be made or that any use will not infringe any patents. 7

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